The covalent attachment of ubiquitin to proteins (known as ubiquitination or ubiquitylation) plays a fundamental role in regulating diverse cell processes including protein degradation, membrane protein trafficking, protein localization, enzyme activation, and DNA repair (reviewed in 
). Covalent attachment of ubiquitin to a substrate occurs through a sequential activation and conjugation of ubiquitin to a target protein by a series of three reactions 
. This is initiated by the ATP-dependent covalent attachment of the ubiquitin molecule to the active site cysteine of the ubiquitin activating enzyme (E1). The ubiquitin molecule is then transferred to via a transesterificaiton reaction to the active site cysteine on an ubiquitin-conjugating enzyme (E2). Subsequently, the E2 interacts directly with an ubiquitin ligase (E3) which facilitates the transfer of the ubiquitin molecule to the substrate. RING finger (RF) proteins constitute the majority of E3s, and accordingly they are fundamental regulators of many key cellular processes 
. The RF is comprised of ~40–60 amino acids that form a coordination complex with two zinc ions 
. The RF interacts with the E2 to mediate transfer of ubiquitin from the active site of the E2 in most cases to an acceptor lysine on target proteins or the growing end of ubiquitin chains. The E3 activity of RF proteins is regulated by covalent modifications of the substrate (e.g.
, phosphorylation) 
, homo- and heterodimerization mediated by the RF domains (e.g.,
Mdm2 homodimers, Mdm2/MdmX heterodimers, and BARD1/BRCA1 heterodimers) 
, covalent modification of the RF protein (e.g.,
phosphorylation and sumoylation) 
, and interaction with non-RF proteins (e.g.,
MAGE proteins) 
Cbl proteins are RF E3s that negatively regulate signaling by many tyrosine kinases (e.g.,
EGFR, Met, and Src) and tyrosine kinase-dependent pathways (e.g.
, T-cell receptor). There are three mammalian Cbl proteins: Cbl (a.k.a., c-Cbl, Cbl2, and RNF55), Cbl-b (a.k.a., RNF56), and Cbl-c (a.k.a., Cbl-3, Cbl-SL, and RNF57) 
(We have used the HUGO nomenclature for the Cbl proteins. The nomenclature is as follows: Cbl refers to the first mammalian family member identified (a.k.a. c-Cbl, Cbl2, and RNF55); Cbl-b refers to the second mammalian Cbl protein identified (a.k.a. RNF56); Cbl-c refers to the third Cbl protein identified (a.k.a. Cbl-3, Cbl-SL, RNF57)). Cbl proteins have a highly conserved N-terminus consisting of a tyrosine kinase binding (TKB) domain that binds to specific phosphorylated tyrosines on substrates, a catalytic RF domain, and an alpha helical linker region separating the TKB and RF domains 
. The C-termini of the proteins are less highly conserved, although all three mammalian Cbl proteins have a proline rich (PR) region that mediates interaction with SH3-domain containing proteins. Cbl and Cbl-b have ubiquitin associated (UBA) domains at their C-termini that mediate homodimerization or ubiquitin binding, respectively. The E3 activity of the Cbl proteins is negatively regulated by the N-terminus of the proteins and activity is increased upon phosphorylation of a tyrosine in the linker region 
While Cbl and Cbl-b have been well-studied and characterized, less is known about Cbl-c. Cbl-c is expressed exclusively in epithelial cells while Cbl and Cbl-b are widely expressed in mammalian tissues 
. Like Cbl and Cbl-b, the N-terminus of Cbl-c is composed of the highly conserved TKB, RF, and linker region 
. The C-terminus of the Cbl-c protein diverges from the other two Cbl proteins by having a shorter PR domain and lacking a UBA domain. Cbl-c, like Cbl and Cbl-b, is a functional E3 that can ubiquitinate and downregulate the EGFR, v-Src, and RET in cells 
. Mice null for Cbl and Cbl-b have clear immunological and hematological defects that help to define their physiological roles, but mice null for Cbl-c are viable and fertile with no clear abnormality 
. Thus the physiological role of Cbl-c is not clear.
To elucidate the function of Cbl-c, we sought to identify Cbl-c interacting proteins utilizing a yeast two-hybrid screen and identified Hic-5 (a.k.a., TGFb1I1, ARA55) 
as a binding partner. Hic-5 is a member of the LIM domain containing paxillin family and the mRNA is widely expressed in normal tissues including epithelial tissues such as the lung, liver, and kidney and the mRNA is expressed in normal epithelial cells in culture 
. Interestingly, the mRNA is low or absent in transformed cells from hematopoietic and epithelial malignancies 
. Hic-5 has been shown to be involved in many events involving cytoskeletal rearrangements, including focal adhesion formation, nuclear/cytoplasmic transport, cell migration, myotube formation, and epithelial to mesenchymal transition 
. The Cbl-c/Hic-5 interaction is mediated by the RF of Cbl-c and the second LIM domain of Hic-5. Importantly, we established that Hic-5 enhances the E3 activity of Cbl-c both in vitro
and in cells. Thus we have identified a novel interaction between two distinct zinc coordinating structures leading to enhancement of the Cbl-c RF E3 activity.